The minimal oxidation of low density lipoproteins (LDL) in the artery wall is thought to initiate the atherogenic process by contributing to macrophage foam cell formation. High density lipoproteins (HDL) promote the efflux of excess cholesterol from macrophage foam cells thus reversing the atherosclerotic process. The enzyme lecithin cholesterol acyltransferase (LCAT) esterifies cholesterol on HDL and thus facilitates the net efflux of cholesterol from foam cells. As a result, LCAT plays an important protective function in reversing atheromatous lesions. Recent studies suggest that HDL may serve a beneficial function by accepting oxidized lipids from minimally oxidized LDL thus inhibiting early atherogenesis. However, lipophilic oxidation products have been found to produce a dramatic inhibition of LCAT activity suggesting the transfer of oxidized lipids from minimally oxidized LDL to HDL may impair HDL function and exacerbate developing atherosclerotic lesions. The aims of this proposal are to 1) identify the oxidized lipids that inhibit LCAT activity, 2) elucidate the underlying mechanism of LCAT impairment, and 3) establish whether the HDL-associated enzyme, paraoxonase (PON), can protect LCAT activity from specific molecular species of lipid peroxides. Preliminary results establish that physiological concentrations of phospholipid hydroperoxides (PL-OOH) are potent inhibitors of LCAT activity and that PON may play a protective role. A major goal of the present proposal will be to define natural boundaries wherein HDL can accept and degrade PL-OOH without sacrificing LCAT activity. It is hypothesized that the first step in HDL degrading PL-OOH involves the transfer of PL-OOH from LDL to HDL; if allowed to accumulate in HDL, PL-OOH directly inactivate the LCAT enzyme. A sensitive HPLC equipped with an on-line, post-column chemiluminescence detection system will be used to examine the transfer of PL-OOH from LDL to HDL. State-of-the-art Electrospray Mass Spectroscopy and protein sequencing techniques will be employed to identify the oxidation products forming specific amino acid adducts directly involved in LCAT inactivation. Site directed mutagenesis will be performed to genetically engineer an active LCAT enzyme resistant to the inhibitory effects of PL-OOH; thus, the underlying mechanism of LCAT impairment will be definitively established. Purified preparations PL-OOH, LCAT and PON enzymes will be used to define the capacity of HDL to accept/degrade PL-OOH without sacrificing LCAT activity. The proposed studies will greatly advance our understanding of the atherogenic process by defining deleterious effects of oxidized lipids on HDL cholesterol transport. Moreover, the research will uncover novel mechanisms for preserving HDL/LCAT function that can be utilized therapeutically to fight the onset of cardiovascular disease.